Purification, some properties and nucleotide sequence of 5‐carboxymethyl‐2‐hydroxymuconate isomerase of Escherichia coli C

FEBS Letters

Roper, David I.¹  Cooper, Ronald A.¹ 

[1] Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK

关键词: 5-Carboxymethyl-2-hydroxymuconate isomerase;    Protein purification;    N-terminal sequence;    Gene sequence;    Derived primary structure;    Escherichia coli C;   

DOI  :  10.1016/0014-5793(90)81507-K

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

As part of an investigation into the evolution of catabolic pathway enzymes a cloned gene encoding the Escherichia coli C 5-carboxymethyl-2-hydroxymuconate (CHM) isomerase, an enzyme of the homoprotocatechuate catabolic pathway, was used to produce large amounts of the protein. The isomerase was purified to homogeneity and some of its properties determined. The reaction occurred optimally at pH 7.6 and the specificity constant was 5.8 × 10⁵ M⁻¹·s⁻¹ with CHM and 6.0 × 10² M⁻¹·s⁻¹ with 2-hydroxyhepta-2,4-diene-1,7-dioate, the substrate of a second isomerase in the pathway. The pure protein showed one type of subunit of M _r 14 000 whilst the molecular mass of the native enzyme was 30 000, suggesting that it was a dimer of identical subunits. The first 19 N-terminal amino acids were sequenced and the data used to confirm that the open reading frame of 378 bp, identified from the nucleotide sequence, encoded the CHM isomerase.

【 授权许可】

Unknown